Almost all organisms on Earth share the basic metabolic pathways that support anaerobic metabolism, and yet many organisms, including most vertebrates, cannot survive for long without molecular oxygen. Embryos of the annual killifish Austrofundulus limnaeus are an excellent model for investigating the mechanistic basis of anoxia-tolerance and anoxia-sensitivity in vertebrates. Embryos of A. limnaeus undergo a unique period of developmental dormancy called diapause. Recent evidence suggests that embryos of A. limnaeus have some very unique physiological adaptations that are associated with tolerance of long-term anoxia. Both dormant and actively developing embryos of A. limnaeus can survive for months without oxygen at 250C. Embryos of A. limnaeus display a massive depletion of ATP during the initial hours of anoxic exposure and lose their mitochondrial membrane potential during this same time frame. These two events are typically associated with cell death in other vertebrate cells, but embryos of A. limnaeus quickly recover from these drastic changes in mitochondrial physiology and energetics. These observations imply that cells of A. limnaeus embryos have some extraordinary characteristics compared to other vertebrates, and even to other vertebrates that exhibit substantial tolerance of anoxia. I will identify the metabolic pathways that support anoxia tolerance in isolated cells of A. limnaeus, assess mitochondrial function and energetics during anoxia and recovery from anoxia, and test the hypothesis that an alternate metabolic pathways supported by the enzyme phosphoenolpyruvate carboxykinase is critical for the survival of anoxia. By using the integrative approaches outlined in the proposal we can hopefully create a more complete picture of the cellular physiology of anoxia-tolerance in the cells if this exceptional vertebrate extremophile. PUBLIC HEALTH RELEVANCE: Heart disease and stroke are responsible for the vast majority of deaths in the developed world. The extreme sensitivity of human heart and brain tissue to lack of oxygen is poorly understood at the cellular level. By understanding the cellular mechanisms that support extreme anoxia tolerance in embryos of the annual killifish, Austrofundulus limnaeus, we may be able to develop treatments to mediate or prevent the damaging effects of heart attacks and strokes to humans.